Calcium channel blocking polypeptide from agelenopsis aperta and therapeutic methods employing it

ABSTRACT

PCT No. PCT/US93/05392 Sec. 371 Date Apr. 27, 1995 Sec. 102(e) Date Apr. 27, 1995 PCT Filed Jun. 10, 1993 PCT Pub. No. WO94/02511 PCT Pub. Date Feb. 3, 1994A polypeptide isolated from the venom of the Agelenopsis aperta spider blocks calcium channels in cells of various organisms and is useful in blocking such calcium channels in cells per se, in the treatment of calcium channel-mediated diseases and conditions, and in the control of invertebrate pests.

This application is a continuation of application Ser. No. 07/919,538,filed 27 Jul. 1992 and now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to a polypeptide found in the venom of theAgelenopsis aperta spider and to a polypeptide having substantially thesame amino acid sequence and substantially the same activity as saidpolypeptide. The polypeptides and the pharmaceutically acceptable saltsthereof block calcium channels in cells including neuronal and musclecells of various organisms including invertebrates and vertebrates. Thisinvention also relates to the use of said polypeptides and their saltsin blocking calcium channels in cells such as cells in the nervous andmuscular system of an organism, per se, and in the treatment of calciumchannel mediated diseases and conditions in a mammal. Further, thisinvention relates to compositions comprising said polypeptides and saltsthereof.

Compounds which are calcium antagonists have a variety of utilities.Calcium antagonists can find clinical application in the treatment ofsuch conditions as angina, hypertension, cardiomyopathies,supraventricular arrhythmias, aesophogeal achalasia, premature labor andRaynaud's disease among others. See W. G. Nayler, Calcium Antagonists,Academic Press, Harcourt Brace Jovanovich Publishers, New York, N.Y.1988, the teachings of which are incorporated herein by reference.Further, such compounds are useful in the study of the physiology ofcells such as neuronal and muscle cells.

Other polypeptides isolated from Agelenopsis aperta are disclosed inU.S. Pat. No. 5,122,596.

SUMMARY OF THE INVENTION

This invention concerns a polypeptide found in the venom of theAgelenopsis aperta spider. The polypeptide of this invention and thefraction in which it is present according to this invention are asfollows.

Agelenopsis peptide J₂ has the following amino acid sequence, SEQ ID NO:1.

H₂N-Glu-Ala-Cys-Ala-Gly-Ala-Tyr-Lys-Ser-Cys-Asp-Lys-Val-Lys-Cys-Cys-His-Asp-Arg-Arg-Cys-Arg-Cys-Asn-lle-Ala-Met-Asp-Asn-Cys-Val-Cys-Lys-Leu-Phe-Tyr-Cys-Glu-Leu-Phe-Gly-Thr-Cys-Asp-Arg-Leu-Lys-Pro

The polypeptide of this invention blocks calcium channels in cells.Accordingly, this polypeptide is useful in blocking calcium channels incells, per se. This polypeptide is also useful in the control ofinvertebrate pests and in the treatment of diseases and conditions in amammal mediated by calcium channel function in cells.

Also within the scope of this invention are polypeptides which havesubstantially the same amino acid sequence and substantially the samecalcium channel blocking activity as the polypeptide described above.

This invention also concerns pharmaceutical compositions comprising saidpolypeptides and methods of administering said polypeptides.

DETAILED DESCRIPTION OF THE INVENTION

Venom is obtained from the Agelenopsis aperta spider through the processof milking by electrical stimulation according to standard methods wellknown to those skilled in the art. It is preferred that the methodemployed is one which safeguards against contamination of the wholevenom by abdominal regurgitant or hemolymph. Such methods are well knownto those skilled in the art. The whole venom so obtained is stored in afrozen state at about -78° C. until used for purification as describedbelow. Purification of the constituents from the whole venom isaccomplished by reverse phase high performance liquid chromatography(HPLC) on a variety of preparative and semi-preparative columns such asC-4 and C-18 Vydace® columns (Rainin Instrument Co. Inc., Mack Road,Woburn Mass. 01801 ). Peak detection is carried out monochromatically at220-230 nm. Further analysis of the fractions can be accomplished with,for example, polychrome UV data collected with a Waters 990 diode arraydetector (Millipore Corporation, Waters Chromatography Division, 34Maple Street, Milford, Mass. 01757). The fractions from the columns arecollected by known methods such as through the use of an ISCO/"FOXY"fraction collector and an ISCO 2159 peak detector (ISCO, 4700 Superior,Lincoln, Nebr. 68504). The fractions are collected in appropriatelysized vessels such as sterile polyethylene laboratoryware. Concentrationof the fractions is then accomplished by lyophilization from the eluantfollowed by lyophilization from water. Purity of the resultingconstituent fractions then can be determined by chromatographic analysisusing an analytical column with a gradient system which is moreisocratic than the system used in the final purification of thefractions.

The polypeptide of the invention can be sequenced according to knownmethods. A general strategy for determining the primary structureincludes, for example, the following steps. 1) Reduction andS-pyridylation of disulfide-bridged cysteine residues to enhancesubstrate susceptability to enzymatic attack. 2) Controlled cleavage ofthe peptide through single or multi-step enzymatic digestion. 3)Isolation and purification of peptide fragments via reverse phase highperformance liquid chromatography (HPLC). 4) Characterization of peptidefragments through N-terminal sequencing and ion-spray mass spectrometry.

S-pyridylethylation of cysteine residues of the polypeptides under studycan be performed, for example, in solution followed by amino acidsequencing of the polypeptides. One such procedure forS-pyridylethylation can be accomplished as described below.

About 1 to 10 μg of polypeptide is dissolved or diluted in up to 50 μlof a buffer prepared by mixing 1 part 1M TrisHCl, pH 8.5, containing 4mM EDTA and 3 parts 8M guanidine-HCl. 2.5 μl of 10% aqueous2-mercaptoethanol is added and the mixture is incubated at roomtemperature in the dark under argon for two hours. After incubation, 2μl of 4-vinylpyridine (fresh reagent stored under argon at -20° C.) isadded and the mixture is incubated for another two hours at roomtemperature in the dark under argon. The mixture is then desalted,preferably by chromatography on a short, reverse phase column. Therecovered alkylated polypeptide is then sequenced according to knownmethods.

Given the benefit of the disclosure herein with respect to the peptidepresent in fraction J₂ of venom from Agelenopsis aperta, it is nowpossible to obtain said peptide by methods other than throughisolation/purification from whole venom. The polypeptides of thisinvention can be produced using recombinant DNA techniques through thecloning of a coding sequence for said polypeptides or portions thereof.For example, hybridization probes which take advantage of the now knownamino acid sequence information of said polypeptide can be employedaccording to methods well known to those skilled in the art to clone acoding sequence for the entire polypeptide. A combination of recombinantDNA techniques and in vitro protein synthesis can also be employed toproduce the polypeptides of this invention. Such in vitro proteinsynthesis methods include, but are not limited to, use of an ABI 430Asolid phase peptide synthesizer (Applied Biosystems, Inc., 850 LincolnCenter Drive, Foster City, Calif. 94404) employing standard Merrifieldchemistry or other solid phase chemistries well known to those skilledin the art.

It is well known in the art that certain amino acid substitutions can bemade in polypeptides which do not affect, or do not substantiallyaffect, the function of said polypeptides. The exact substitutions whichare possible vary from polypeptide to polypeptide. Determination ofpermissible substitutions is accomplished according to procedures wellknown to those skilled in the art. Thus, all polypeptides havingsubstantially the same amino acid sequence and substantially the samecalcium channel blocking activity are within the scope of thisinvention.

The polypeptides of this invention block calcium channels present in avariety of cells such as cells in the nervous and muscular system ofinvertebrates and vertebrates.

The ability of the polypeptides of this invention to block calciumchannels is demonstrated by the following procedure. Cerebellar granulecells are prepared from the cerebellum of 8 day old rats (Wilkin et al.,Brain Res, 115, 181-199, 1976). Squares (1 cm²) of Aclar (ProplasticsInc., 5033 Industrial Ave., Wall, N.J. 07719) are coated withpoly-L-lysine and placed in 12-well dishes that contain 1 ml of EaglesBasal Medium. The cells are dissociated and aliquots containing 6.25×10⁶cells are added to each well containing the squares of Aclar.Cytosine-beta-D-arabino furanoside (final concentration 10 μM) is added24 hours after plating. The cells are used for fura2 analysis at 6, 7and 8 days of culture. The cells (attached to the Aclar squares) aretransferred to 12 well dishes containing 1 ml of 2 μM fura2/AM(Molecular Probes Inc., Eugene, Oreg. 97402) in HEPES buffer (containing0.01% bovine serum albumin, 0.01% dextrose, pH 7.4, magnesium-free). Thecells are incubated for 40 minutes at 37° C.; the fura2/AM-containingbuffer is removed and replaced with 1 ml of the same buffer withoutfura2/AM. To a quartz cuvette is added 2.0 ml of prewarmed (37° C.)buffer. The cells on the Aclar are placed in the cuvette and the cuvetteis inserted in a thermostatted (37° C.) holder equipped with a magneticstirrer and the fluorescence is measured with a fluorescencespectrophotometer (Biomedical Instrument Group, University ofPennsylvania). The fluorescence signal is allowed to stabilize for abouttwo minutes. Then 5-20 μl of a stock solution of the compound understudy in phosphate buffered saline (PBS, pH 7.4) at appropriateconcentration is added to the cuvette. Calibration of the fluorescentsignals and fura2/AM leakage correction are performed using theestablished procedures of Nemeth et al., J. Biol. Chem., 262, 5188(1987) at the completion of each test. The maximum fluorescence value(Fmax) is determined by addition of ionomycin (35 μM) and the minimumfluorescence value (Fmin) is determined by the subsequent addition ofEGTA (12 mM) to chelate calcium. Employing the foregoing procedure,calcium channel blocking by a subject polypeptide is shown to occur by adecrease in fluorescence upon addition of the subject polypeptide. Thepolypeptide of the invention exhibits low IC₅₀ values, under 200 nm, forblocking calcium channels using this assay. For comparison, two knowncommercial calcium channel antagonists, Nifedipine and Verapamil, haveIC₅₀ values of 33 nm and 4800 nm, respectively.

The polypeptides of this invention are useful as calcium channelblockers in cells, per se. As such, these polypeptides are also usefulin the control of invertebrate pests and in the treatment of diseasesand conditions mediated by calcium channels function in cells in amammal such as angina, hypertension, cardiomyopathies, supraventriculararrhythmias, aesophogeal achalasia, premature labor and Raynaud'sdisease. Further, these polypeptides are useful in the study of thephysiology of cells including, but not limited to, cells of the nervousand muscular system.

Also within the scope of this invention are the pharmaceuticallyacceptable salts of the polypeptides of this invention. Such salts areformed by methods well known to those skilled in the art. For example,base salts of the polypeptides can be prepared according to conventionalmethods.

When a polypeptide of this invention is to be administered to a mammal,it can be administered alone or in combination with pharmaceuticallyacceptable carriers or diluents in a pharmaceutical compositionaccording to standard pharmaceutical practice. The polypeptides can beadministered orally or parenterally with the parenteral route ofadministration being preferred for polypeptides. Parenteraladministration includes intravenous, intramuscular, intraperitoneal,subcutaneous and topical administration.

For oral use of a polypeptide of this invention, the compound can beadministered, for example, in the form of tablets or capsules, or as anaqueous solution or suspension. In the case of tablets for oral use,carriers which are commonly used include lactose and corn starch, andlubricating agents, such as magnesium stearate, are commonly added. Fororal administration in capsule form, useful diluents are lactose anddried corn starch. When aqueous suspensions are required for oral use,the active ingredient is combined with emulsifying and suspendingagents. If desired, certain sweetening and/or flavoring agents can beadded.

For intramuscular, intraperitoneal, subcutaneous and intravenous use,sterile solutions of the active ingredient are usually prepared, and thepH of the solutions should be suitably adjusted and buffered. Forintravenous use, the total concentration of solutes should be controlledto render the preparation isotonic.

When a polypeptide or salt thereof of this invention is used in a humansubject, the daily dosage will normally be determined by the prescribingphysician. Moreover, the dosage will vary according to the age, weightand response of the individual patient, as well as the severity of thepatient's symptoms and the potency of the particular compound beingadministered.

When a polypeptide or salt thereof of this invention is used in controlof invertebrate pests, said polypeptide is administered to saidinvertebrate directly or provided to the environment of saidinvertebrate. For example, a compound of this invention can be sprayedas a solution onto said invertebrate. The amount of compound necessaryfor control of said invertebrate will vary according to the invertebrateand environmental conditions and will be determined by the personapplying the compound.

When a polypeptide or salt thereof of this invention is used in thephysiological study of cells, said polypeptide is administered to thecells according to methods well known to those skilled in the art. Forexample, said polypeptide can be administered to cells in an appropriatephysiological buffer. An appropriate concentration of a polypeptide ofthis invention for use in such studies is 200 μM. However, theconcentration of said polypeptide in such studies may be greater than ormuch less than 200 μM. The amount of the polypeptide administered willbe determined by the person skilled in the art according to well knownmethods.

EXAMPLE B 1

A. Crude Agelenopsis aperta venom (˜40 μl) was applied to a reversedphase HPLC column (VYDAC® C-18, 300 Å, 22×250 mm) operated using abiphasic linear gradient program from 95% A and 5% B to 80% A and 20% Bover 30 minutes, then to 30% A and 70% B over 25 minutes (A=0.1%trifluoroacetic acid and B=CH₃ CN), with detection at 220 nm and a flowrate of 15 ml/minute. The desired fraction was collected from 38.3 to38.7 minutes. Pooled like fractions from individual runs wereconcentrated by lyophilization.

B. The material from the fractionation of step A, above, derived from100 μl of crude venom, was applied to a reversed phase HPLC column(Vydac®, C-18, 300 Å, 22×250 mm) operated using a linear gradientprogram from 77% A and 23% B to 70% A and 30% B over 25 minutes (A =0.1%trifluoroacetic acid and B=CH₃ CN) with detection at 220 nm and a flowrate of 12 ml/minute. The desired fraction was collected from 17.3 to17.7 minutes. Pooled like fractions from individual runs wereconcentrated by lyophilization.

The structure of peptide J₂ was determined and verified by the followingmethods. PTC amino acid analysis was carried out on 1 to 10 nmols intriplicate using the Waters Pico-Tag system. N-terminal sequencing wascarried out on a pulse-liquid sequenator (ABI) both on native and onreduced/pyridylethylated peptide. The primary structure of thepolypeptide was obtained by use of an automated pulse liquid sequencer(Applied Biosystems, model 473A). Mass spectral analysis data wasobtained from a BIO-ION plasma desorption time of flight massspectrometer.

A pyridylethylated derivative of peptide J₂ suitable for N-terminalsequencing was prepared in the following fashion. Peptide J₂ (50 mg) wasdissolved in 10 μl of buffer (1:3 ratio of 1M tris, pH 8.4, 4 μMEDTA-dibasic and 8M guanidine-hydrochloride and was treated with 2 μl ofa 0.454M (10% v/v) solution of 2-mercaptoethanol in buffer and kept for3 hours in the dark at room temperature. The reaction mixture was thentreated with 2 μl of a 0.456M solution of 4-vinylpyridine in buffer andkept at room temperature in the dark for 18 hours. The reaction mixturewas diluted with 90 μl of water and 40 μl of acetonitrile and applied toan HPLC column (Baker WPC-18, 4.6×250 mm) operated using a biphasiclinear gradient program of 80% A and 20% B for 5 minutes followed by 80to 50% A and 29 to 50% B over 30 minutes (A=0.1% trifluoroacetic acid,B=CH₃ CN) with detection at 220 nM and a flow rate of 1.0 ml/minute. Thedesired fraction was collected at 20.8 to 21.3 minutes and wasconcentrated by lyophilization.

The data taken together affirm the structure of peptide J₂ as shownbelow.

SEQ ID NO: 1, 48 residues, 10 cysteines, 5 disulfide bonds.

Calculated mass=5474.4.

Observed mass=5474.

Estimated pl=7.98.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 1                                                  (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 48 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                          (ii) MOLECULE TYPE: protein                                                   (iii) HYPOTHETICAL: NO                                                        (iv) ANTI-SENSE: NO                                                           (vi) ORIGINAL SOURCE:                                                         (A) ORGANISM: Agelenopsis aperta                                              (F) TISSUE TYPE: venom                                                        (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       GluAlaCysAlaGlyAlaTyrLysSerCysAspLysValLysCysCys                              151015                                                                        HisAspArgArgCysArgCysAsnIleAlaMetAspAsnCysValCys                              202530                                                                        LysLeuPheTyrCysGluLeuPheGlyThrCysAspArgLeuLysPro                              354045                                                                        __________________________________________________________________________

We claim:
 1. A substantially pure polypeptide having the amino acidsequence, SEQ ID NO: 1, or a pharmaceutically acceptable salt thereof.2. A method of blocking calcium channels in a cell comprisingadministering to said cell a calcium channel blocking amount of apolypeptide according to claim
 1. 3. A method according to claim 2wherein said cell is in the nervous system of a mammal.